AU680264B2

AU680264B2 - Synthetic peptides and vaccines against parvovirus

Info

Publication number: AU680264B2
Application number: AU70048/94A
Authority: AU
Inventors: Jose Ignacio Casal Alvarez; Kristian Dalsgaard; Joannes Pieter Maria Langeveld; Robert Hans Meloen; Carmen Vela Olmo
Original assignee: Inmunologia y Genetica Aplicada SA
Current assignee: Inmunologia y Genetica Aplicada SA
Priority date: 1993-01-23
Filing date: 1994-01-21
Publication date: 1997-07-24
Anticipated expiration: 2014-01-21
Also published as: NZ259882A; DK0647655T3; EP0647655A1; ATE174929T1; EP0647655B1; DE69415462T2; GR3029727T3; CA2132742A1; WO1994017098A1; AU7004894A; CA2132742C; US5785974A; DE69415462D1

Abstract

The invention discloses synthetic peptides from autonomous parvoviruses, which are capable of inducing neutralizing antibodies against said viruses. These peptides correspond to antigenic sites located in the first 25 amino acids of the amino terminal end of the VP2 proteins of parvoviruses. When these peptides are coupled to carrier proteins or to other immunogenic complexes, they can be used in the formulation of vaccines appropriate to protect animals against the infection caused by said parvoviruses.

Description

SYNTHETIC PEPTIDES AND VACCINES AGAINST PARVOVIRUS FIELD OF THE INVENTION The present invention relates to chemically synthesized viral peptides related to the major antigen (VP2) of the autonomous parvovirus capsid and to assays and vaccines using the said peptides. The said peptides can, for instance, induce antibodies neutralizing for Canine Parvovirus (CPV), mink enteritis virus (MEV) and Porcine Parvovirus (PPV), whence they can be formulated in vaccines and respectively confer .otal protection against CPV, MEV and PPV in dogs, minks and pigs.

BACKGROUND OF THE INVENTION Parvoviruses form a viral family having certain common features (Cotmore et al., Adv. Virus Res. 33:91-174 (1987)). They are the smallest known DNA viruses. Their genome is formed by a single stranded linear DNA molecule having a length of some 5.0 kb enclosed within a proteic capsid, being icosahedral in shape and having a size of approximately 25 nm. Two large open reading frames (ORF) are detected in the viral genome. The left ORF codes for non-structural proteins involved in viral replication whereas the right ORF codes for structural proteins forming the viral capsid, which is constituted by VP1, VP2 and VP3 proteins. The mRNA of both ORFs is polyadenylated and 3'-coterminal. There are approximately copies of VP2 in the viral capsid and approximately copies of VP1 (Wobble et al., Biochemistry 23, 6565-6569, 1984) which may be arranged as either homo- or heterodimers (Paradiso, J. Virol., 46, 94-102, 1983). The VP2 protein contains all the antigenic determinants involved in neutralizing the virus (L6pez de Turiso et al., J. Gen. Virol., 72, 2445-2456 (1991); Langeveld et al., J. Virol., Vol. 67, No. 2, 765-772 (1993); L6pez de 2 Turiso et al., J. Virol., Vol. 66, No. 5, 2748-2753 (1991)).

Autonomous parvoviruses are responsible for a large number of diseases affecting human beings and animals of interest alike and may indeed be fatal because of the tendency of autonomous parvoviruses to replicate in proliferating cerebellum, lymphoid tissue, intestinal epithelium or foetal tissue cells. Among these autonomous parvoviruses are CPV, MEV, PPV, bovine parvovirus (BPV), goose parvovirus (GPV), feline panleukopenia virus (FPLV) and B19 parvovirus which affects human beings.

In this description the autonomous parvoviruses CPV and PPV will be focussed on as an example. Some of the main features of both parvoviruses are summarised hereinbelow.

Canine Parvovirus (CPV) Within the genus of autonomous parvoviruses, CPV is a member of the feline parvovirus subgroup. The other members of this subgroup, very related each other, are feline panleukopenia virus (FPLV) and MEV. CPV causes severe enteritis in dogs of all ages and myocarditis in puppies less than 12 weeks old. CPV was first isolated in 1978 (Burtonboy, G. et al., Arch. Virol. 61:1-11 (1979); Appel et al., Vet. Rec. 105. 156-179, (1979)) and it is believed to have arisen as a natural variant of FPLV or

MEV.

Protein and DNA sequence studies and serologic studies show a large antigenic and genetic homology between CPV, FPLV, MEV and the Raccoon Parvovirus (Tratschin et al., J. Gen Virol. 61:33-41 (1982); Carlsson et al., J.

Virol., 55, 574-582 (1985); Parrish et al., Arch. Virol.

72, 267-278 (1982); Reed et al., J. Virol. 62:266-276 (1988)). Despite this homology they are exquisitely specific in the "in vivo" host, although "in vitro" all viruses replicate in cat kidney cells (Appel et al., Vet.

Rec. 105, 156-179 (1979); Trastschin et al., J. Gen.

30 Vio 72 26-278(192);Reedet Vrol 62:66-76 (18).Dsieti oooyte r xustl i 2 Turiso et al., J. Virol., Vol. 66, No. 5, 2748-2753 (1991)).

Canine Parvovirus (CPV) Within the genus of autonomous parvoviruses, CPV is a member of the feline parvovirus subgroup. The other members of this subgroup, very related each other, are feline pa!1leukopenia virus (FPLV) and MEV. CPV causes severe enteritis in dogs of all ages and myocarditis in puppies less than 12 weeks old. CPV was first isolated in 1978 (Burtonboy, G. et al., Arch. Virol. 61:1-11 (1979); Appel et al., Vet. Rec. 105. 156-179, (1979)) and it is believed to have arisen as a natural variant of FPLV or

MEV.

Virol., 55, 574-582 (1985); Parrish et al., Arch. Virol.

Rec. 105, 156-179 (1979); Trastschin et al., J. Gen.

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Ii~1 3 Virol., 61:33-41, (1982)). The CPV capsid contains two proteins with broadly overlapping amino acid sequences, VP1 (82-84 KDa) and VP2 (67-70 KDa) (Paradiso et al., J. Gen. Virol. 39, 800-807, (1982); Surleraux et al., Arch Virol., 82, 233-240 (1984); J. Gen. Virol. 62, 113-125, (1982)). VP2 in full capsids (holding DNA) is preferentially broken down by proteolytic digestion into 63-67 KDa VP3 (Paradiso et al., Gen. Virol. 39, 800-807 (1982); Surleaux et al. Arch Virol., 82, 233- 240 (1984)) after capsid assembly (Paradiso, J. Virol., 39, 800-807 (1981)). The three-dimensional structure of the CPV capsid is currently known (Tsao et al., Science 251: 1456-1464 (1991)).

Our laboratory has investigated into the immunogenicity of various fragments of the proteins making up the CPV viral capsid, which has resulted in new VP2 protein and VP2 and VP1 peptides based recombinant vaccines being described. These findings are summarised in Spanish Patent Number P9100844, which relates to the expression 20 of VP2 in a recombinant baculovirus system.

Ten antigenic sites in the VP2 sequence and their spatial location upon the capsid surface have been o identified Langeveld et al., J. Virol. 67. No. 2 (1993)).

25 Four potential neutralization sites have been described in CPV. Two such sites were mapped with synthetic Speptides and of these, one is found at the N-terminal S. end and the other at positions 147-163 of VP2 (Rimmelzwaan et al., J. Gen. Virol., 71:2741-2745 (1990). The two other sites were mapped on and about a large protuberant domain on the three-fold axis of symmetry of the viral capsid (Parrish et al., Virology S 166:293-307 (1988)).

The role of the amino terminal end of VP2 in parvovirus is the subject of speculation. This domain is important

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because "in vitro" viral neutralization has been observed when a monoclonal antibody specific to this site is used (L6pez de Turiso et al., J. Gen. Virol., 72:2445-2456 (1991)), it is immunogenic in various animal species (Langeveld et al., J. Virol., Vol. 67, No. 2, 765-772 (1993)) and it is involved in the attachment of the virus to the cell.

The peptide vaccines described herein are directed against part of this site. The knowledge of antigenic sites in VP2 led to a collection of synthetic peptides of potential interest in vaccine formulation being designed. Synthetic peptides including amino acids of the amino antibodies in test animals. This domain is therefore an attractive candidate for its inclusion in a synthetic peptide vaccine.

Conventional CPV vaccines based upon live of inactivated virus and recombinant protein-based vaccines are currently available but there are no synthetic vaccines capable of inducing antibodies neutralizing CPV and effectively protecting dogs from CPV infection. These vaccines would provide manifold advantages, being not only economic but operative.

Vaccines based on synthetic peptides are safe, because they eliminate the risks stemming from the handling and diffusion of the infectious agents inherent in conventional vaccines, are easy to produce, are highly reproducible and are very stable. In the case of CPV, another significant advantage of a synthetic vaccine is its potential to prevent vaccinal problems arising ftom the natural immunity in young puppies. Conventional CPV vaccines do not ensure complete prbtection in puppies less than 10 weeks old, for such have maternal antibodies which eliminate the vaccine. The data known heretofore point to the fact that among the general population of anti-virus antibodies in infected ~1 i j 3' P:~p~J 4)~ 0n II- I j: t animals, anti-peptide antibodies are not present in large quantities, if at all. If antibodies induced by synthetic peptides are not predominant "in vivo" the peptidic vaccine would allow the immune response in the neonate at an earlier age than under current conditions.

Now therefore, an object of the present invention lies in new chemically synthesized peptides which include all or part of the terminal amino end of VP2 in CPV capable of inducing antibodies and neutralizing CPV.

Given the close homology (in excess of 98%) between CPV, MEV and FPLV it is very likely that an agent capable of inducing protection in dogs will have this same effect in other hosts, such as cats and minks.

15 This effect will, for instance, be demonstrated in ^minks. Consequently, these new peptides may be used I for obtaining immunogenic compositions and in formulating new synthetic vaccines capable of protecting dogs and, alternatively, minks and cats, 20 respectively from CPV, MEV and FPLV infections. The said immunogenic compositions and vaccines are a further object of this invention.

The term "synthetic peptides" as used herein may refer to chemically synthesised peptides or peptides obtained 25 from intracellular synthesis using recombinant techniques which are well known to those of skill in the art. Preferably, the synthetic peptides of the invention are obtained by chemical synthesis.

Porcine Parvovirus (PPV) PPV causes reproductive failures in pigs, leading to death and foetal mmumification, abortions and other reproductive failures in pregnant pigs (Joo Johnson, Veterinary Bulletin 46, 653-660 (1976); Mengeling, J.

Am. Vet. Med. Assoc. 172, 1291-1294 (1978)). PPV o\ contains a single-stranded DNA molecule with I Vi' 'I 1-I- C S o ,o.

approximately 5000 nucleotides (Mollitor et al., Virology 137, 241-254 (1984)). The full sequence of the PPV genome has been described by our group (Ranz et al., J. Gen. Virol. 70, 2541-2553 (1989)). Four specific virus proteins have been described: three proteins forming the capsid (VP1, VP2 and VP3 with respective molecular weights fo 83000, 64000 and 60000 dalton) and a non-structural protein NS1. Of the three structural proteins, the Vp2 protein in PPV, just as VP2 in CPV, contains probably all the antigenic determinants involved in PPV neutralization.

There currently exist vaccines which provide porcine parvovirus protection based upon traditional inactivation methods with chemical agents and/or search of attenuated mutants for the virus. All previous attempts at producing new vaccines using recombinant proteins produced in prokaryotic microorganisms (v.gr.

E.coli) have however failed.

In recent years, our laboratory has been working on PPV molecular biology Ranz et al., J. Gen. Virol.

70, 2541-2553 (1989)); J.I. Casal et al., Virology, 177, 764-767 (1990)). These papers are related to the knowledge of viral DNA sequences which code for the proteins forming the PPV cpasid. These sequences allowed us to identify the gene coding for VP2 in PPV and manipulate and insert the same in suitable vectors for expressing the same in a baculovirus system, as described in Australian Patent Application No.

15850/92, related to the expression of VP2 in a recombinant baculovirus vector.

Using techniquies resembling those used with CPV, assays were conducted designed to establish and search for atnigenic sites in PPV. The results obtained showed that, as with;CPV, the amino terminal end of VP2 in PPV is a potential neutralization site.

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ft r *a oi *e C 3 -0PrAo C7Vo AV r 6a Therefore, although there are currently conventional and recombinant protein-based PPV vaccines available, there is no synthetic vaccine capable of effectively protecting pigs from PPV infection. It would be useful to have synthetic vaccines incorporating small peptides capable of inducing neutralizing antibodies, which would be both economically and operatively beneficial.

Consequently, a futher object of this invention lies in new synthetic peptides stemming from the animo terminal end of VP2 in PPV, capable of inducing antibodies *t Po t neutralizing PPV, which may be used in the obtention of immunogenic compositions and in formulating new synthetic vaccines capable of protecting pigs from PPV infection.

These synthetic peptides are based upon the immunogenic properties of the amino end of VP2 the major protein inl PPV. The immunogenic compositions and vaccines which incorporate such peptides are a further object of this invention.

Similarly, because of the structural similarity between the amino terminal ends of the VP2 proteins in all autonomous parvoviruses and the fact that in all animals tested in this invention (rabbits, hamsters, dogs, minks and pigs) consistently good titres of neutralizing antibodies were obtained, it follows that synthetic peptides found at similar positions, i.e. at the amino terminal end of the relevant VP2 proteins, could also be used to vaccinate humans against B19 Parvovirus, cattle i against Bovine Parvovirus (BPV), geese against Goose Parvovirus (GPV) and, in general, against any other autonomous Parvovirus.

BRIEF DESCRIPTION OF THE FIGURES Figure 1: shows the results obtained in establishing the titre of anti-CPV antibodies present in the serum of dogs immunized with the peptides of this invention (dogs 3029-Arand 3030-0-) and in a sentry dog (3025 using a specific i virus ELISA (Enzymatic Immunoassay); Figure 2: shows the results obtained in establishing the titre of anti-peptide antibodies (1L15 and 7L15) present in the serum of immunized dogs (3029 and 3030) and in the sentry dog (3025), using a specific peptide ELISA, according to the following key: :anti-1L15 titre in sentry dog (3025 serum I INMUNOLOGIA Y GENETICA APLICADA, S.A.,

BY:

Carme Vela Olmo (Manager Director) ka' ii. 1

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Fic SA- :anti-1L15 titre in immunized dog (3029 serum -:anti-lL15 titre in immunized dog (3030 serum :anti-7L15 titre in sentry dog (3025 serum :anti-7L15 titre in immunized dog (3029 serum :anti-7L15 titre in immunized dog (3030 serum gure 3: shows the results obtained in establishing the titre of antibodies neutralizing CPV in the serum of sentry dog (3025 and dogs immunized with peptides of this invention (3029--and 3030- jure 4: shows the results obtained in establishing the presence of CPV in the faeces of immunized dogs (3029-A-and 3030-6) and in the faeces of sentry dog (3025-M-) using a specific viral antigen ELISA.

Fic DETAILED DESCRIPTION OF THE INVENTION The invention provides new peptides useful in preparing suitable synthetic vaccines for protecting animals from infections caused by autonomous parvoviruses, such as CPV, MEV, FPLV and PPV. The new vaccines contain at least a chemically synthesized peptide originating in the amino terminal region of the VP2 protein in CPV or the VP2 protein in PPV. As used herein, terminal amino region should be construed as the region comprising at least the first twenty-five aminc acids of the VP2 protein.

1.

It, haso been found by PEPSCAN analysis (Geysen H.M. et Sal., Proc. Natl. Acad. Sci., USA, 81:3998-4002 (1984)) that there are ten antigenic sites in the VP2 of CPV

B

1~ i:"i a 9 (Langeveld et al., cited supra). Other authors have described other antigenic sites (Rimmelzwaan et al., and Parrish et al., (1988) cited supra). Based upon all the sites described a collection of peptides was synthesized to cover such sites. On investigating the immunogenicity of all the synthesized peptides it was found that only the peptides originating in the first twenty-five amino acids at the amino terminal end of the VP2 in CPV, coupled to KLH [Keyhole Limpet Hemocyanin] were capable of inducing neutralizing antisera in rabbits. The said peptides are capable of protecting dogs from the pathogenic effects of CPV infection.

The peptides capable of inducing antibodies neutralizing CPV, provided by this invention, are contained within Identified Sequence No. 1 (ID. SEQ. No. 1) [see the section regarding the Sequence List].

This invention includes all the peptides comprised within ID. SEQ. No. 1 or resulting from the substitution cf some of the amino acids shown in ID. SEQ. No. 1 with other functionally equivalent amino acids, or from the deletion or insertion of some such amino acids provided the resultant peptides are capable of inducing antibodies neutralizing CPV. Synthetic peptides with a length of between 6 and 20 amino acids are preferred for practical reasons.

Generally speaking, the peptides of the invention shall preferably be soluble in order that they may be coupled to suitable carrier proteins enhancing their immunogenic character, such as the KLH protein or any other protein used habitually in this field.

To obtain and select the synthetic peptides of this invention the following steps were basically taken: a) Chemically synthesizing the peptides corresponding to the most immunogenic regions of the VP2 protein in CPV.

(I i 1 1 b) Coupling such peptides to a carrier protein.

c) Selecting the peptides inducing an antibody response in rabbits capable of neutralizing CPV.

d) Immunizing dogs with the peptides selected above and holding and carrying out a challenge test with virulent virus to check protection of dogs from infection.

These steps will be described hereinafter in greater detail. In a specific embodiment (Example 1) twenty-one pentadecapeptides were synthesized using Fmoc chemistry (Fields C. G. et al., Peptide Res., 4:95-101 (1991)) which briefly comprises: preparing a resin (solid phase) incorporating the amino acid of the C-terminal end with its amino end protected by a suitable protector group such as Fmoc (9fluorophenyl-methyl-oxy-carbonyl) or the like (terbutoxycarbonyl, benzyloxycarbonyl, p-toluenesulphonyl, triphenylmethyl, or o-nitrophenylsulphenyl); successively incorporating in a suitable order the 'emaining amino acids with their amino groups duly protected; leaving the synthesized peptide without protection and separating the same from the resin by suitable treatment; and purifying the chemically synthesized peptide.

The twenty-one synthesized peptides were coupled (Example where possible, to the carrier KLH protein and used 't immunize rabbits (Example With immunization over Y it was found that the 1L1S, 2L15, 3L15, 4L15, 5L15, 6L15, 7L15, 8L15 and 9L15 peptides were capable of inducing antibodies neutralizing CPV.

It was additionally found that dogs vaccinated with a mixture of the 1L15 and 7L15 peptides developed a high titre of antibodies neutralizing the virus and were protected from later infection with virulent virus T s w deal!naseii moiet(xml )tet-n 8 I (Example Based upon these results i can be stated that'he peptides provided by this invention can be used in formulating new synthetic vaccines in order to protect animals from CPV infection. Furthermore, due to the structural resemblance and the quasi-identity of the sequences of the first twenty-five amino acids of the terminal amino ends of the VP2 proteins in CPV, MEV and FPLV, it has been shown that peptides comprised within ID. SEQ. No. 1 are capable of inducing antibodies "in vivo" neutralizing MEV and can therefore be used in formulating vaccines capable of protecting minks from MEV infection. Indeed, it has been found that minks vaccinated with a mixture of the 1L15 and 7L15 peptides were protected from later infection with virulent virus (Example It can be assumed from this test confirmation with MEV that the peptides included withini f ID. SEQ. No. 1 can induce FPLV protection in cats due to the structural homology between CPV, MEV and FPLV, which Samll belong to the same parvovirus subgroup.

Furthermore, given the structural homology there is between all the autonomous parvoviruses it was assumed that using the same procedure similar results could be SFI found in .other parvoviruses, such as PPV, BPV or the human Bi9 parvovirus. This demonstration was, for instance, made in PPV.

2. PPV )I This invention also provides new peptides useful in the production of suitable synthetic vaccines for protecting pigs from PPV infection. These chemically synthesized peptides originate in the terminal amino region of the VP2 protein in PPV. The immunogenicity of the VP2 in PPV was first investigated using a strategy similar to that followed in the case of the VP2 in CPV. The sequence was analyzed by PEPSCAN and a collection of peptides located ll elog t thesam pavovrus ubgoup Frthrmor, gventhe trutura 'hmolgy tereis 15 i 12 at the identified antigenic regions was synthesized.

These peptides, coupled to KLH, were used to immunize rabbits and it was again found that only the peptides lying in the amino terminal region were capable of inducing antibodies neutralizing PPV and hence protecting pigs from the pathogenic effects of PPV infection. These synthetic peptides are included in ID. SEQ. No. 2.

This invention includes all peptides comprised within ID.

SEQ. No. 2 or resulting from the substitution of some of the amino acids shown in ID. SEQ. No. 2 with other functionally equivalent amino acids or the deletion or insertion of some such amino acids, provided that the resultant peptides are capable of inducing antibodies neutralizing PPV. Synthetic peptides with a length of between,6 and 20 amino acids are preferred for practical reasons.

Generally speaking, the peptide. of the invention shall preferably be soluble in order that they may be coupled to suitable carrier proteins enhancing their immunogenic character, such as the KLH protein.

To obtain and select the synthetic peptides of this invention the following steps were basically followed: a) Chemically synthesizing the peptides corresponding to the most immunogenic regions of the VP2 protein in PPV.

b) Coupling such peptides to a carrier protein.

c) Selecting the peptides inducing an antibody response in rabbits capable neutralizing PPV.

d) Immunizing pigs with the peptides selected above.

These steps will be described hereinafter in greater detail. In a specific embodiment (Example 2) seventeen i solid phase peptides were synthesized using Fmoc V" chemistry, as mentioned before in relation to synthetic iCPV ptes.

The seventeen peptides synthesized were coupled to the il I 1 j 1

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Fg -1 13 carrier KLH protein (Example 3) and were used to immunize rabbits (Example With immunization over it was found that the peptides designated ILl5, 5L16, 6L15, 8L15, 10L16 and 13L16 were capable of inducing antibodies neutralizing PPV (Table Based upon these results it can be stated that such peptides can be used in formulating new synthetic vaccines in order to protect animals from PPV infection.

3. Immunogenic compositions and vaccines Immunogenic compositions can be prepared taking the peptides provided by this invention which contain the said peptides in immunogenic form, whence they can be used to formulate vaccines capable of protecting dogs, cats and minks respectively from CPV, FPLV and MEV infection (to which end peptides comprised within ID.

SEQ. No. 1 will be used) and to formulate vaccinesR capable of protecting pigs from PPV infection (to which end peptides comprised within ID. SEQ. No. 2 will be used). The said immunogenic compositions can be prepared by coupling at least one of the peptides of this invention to a suitable carrier protein or multimeric structure. PCT Patent application published with number WO 90/11298 contains several references relating to coupling methods and carrier proteins which may be used.

In a preferred embodiment of this invention compositions are provided which comprise an immunogenic conjugate of a protein (KLH) and a peptide of this invention. i Alternatively, these compositions can contain an J 30 immunogenic complex obtained by crossing the peptide or

\I

an immunogenic recombinant protein containing one of the peptides of this invention..

This invention also provides vaccines that are characterized by comprising one of the aforesaid immunogenic compositions combined with at least an ^4 aw. AL V 1V V Sd SSIOS, I CI. I.S Jl' n viruses replicate in cat kidney cells (Appel et al., Vet.

Rec. 105, 156-179 (1979); Trastschin et al., J. Gen. 14 Simmunological adjuvant. Such vaccines can be prepared suspending at least one of the peptides of the invention coupled to a suitable carrier protein or multimeric structure, in an immunologically acceptable diluent plus an adjuvant. Additionally, a vaccine can contain a mixture of the peptides of this invention as immunogenic agents. Acceptable immunological diluents used are saline solutions with phosphate, tris or other like saline solutions. The adjuvant used can be alumina gel suspensions alone or combined with other adjuvants used habitually in the formulation of vaccines such as QuilA or the like having a similar power.

DETAILED DESCRIPTION OF AN EMBODIMENT OF THE INVENTION

(EXAMPLES)

Example 1 CHEMICAL SYNTHESIS OF CPV PEPTIDES AND SELECTION OF IMMUNOGENIC PEPTIDES Based upon the data as to the location of B epitopes on the CPV capsid (Langeveld et al., cited supra) the twenty-one peptides shown in Table 1 were synthesized.

ij 1 1 TABLE 1i PEPTIDE CODE SEQUENCE -10L15 *GQVKRDNLAPMSDGA# 3--L15 *DNLAPMSDGAVQPDG# 1L15 *MSDGAVQPDGGQPAV# 2L15 *SDGAVQPDGGQPAVR#

*DGAVQPDGGQPAVJN#

4L15 *GAVQPDGGQPAVRNE# 5L15 *AVQPDGGQPAVRNER# 6115 *VQPDGGQPAVRNERA# 7L15 *QPDGGQPAVRNEPAT# 8L15 *PDGGQPAVRNERATG# 9L15 *DGGQPAVRNERATGS# 11L15 *GQPAVRNERATGSGN# 16L15 *RNERATGSGNGSGGG# 91L15 *AVNGNMALDDIHAQI# 147L17 *NVVLKTVSEDATQPPTK# 172L15 *SLMVALDSNNTMPFT# 283L15 *PJALGLPPFLNSLPQS# 296L15 *QSEGATNFGDIGVQQ# 498L15 *LFVKVAPNLTNEYDP# b49L15 *QQMSINVDNQFNYVP# 570L15 *KIVYEKSQLAPRKLY# 1) In both Table 1 and Table 2: -each letter in the sequence stands for an amino acid designated pursuant to the single letter amino acid nomenclature code (Structural Biochemistry, P.

Louisot, Ed. AC, 1st. Edition (1977), pages 372- 373) the amino terminal end is located to the left of the sequence whereas the carboxy terminal end is located to the right of the sequence; the peptide code shows the position of the B residue of the N-terminal end in the VP2 sequence o I i 16 (before L) and the length of the peptide, stated as a number of amino acid residues (behind L); stands for a Cysteine residue with the acetylated amino group; and denotes that the carboxy group of the carboxy terminal amino acid residue is amidated.

Save for the 157L17 heptadecapeptide, the length of the peptides was of 15 amino acid residues, not including the N-terminal cysteine which was used for coupling. Sixteen peptides were sufficiently soluble to allow their conjugation to KLH whereas the other six displayed only a limited solubility (less than The synthesis of these peptides was carried out using Fmoc chemistry (Saxon Biochemical, Germany), pursuant to the general process mentioned above, on RinkTM resins (Saxon).

Synthesis was carried out either manually in glass vessels or in an Applied Biosystems 430A synthesizer. The sequences are acetylated at the N-terminal end and amidated at the C-terminal.

The peptides were purified using high performance liquid chromatography (HPLC). Quality was analyzed in a Delta PaK 5 p C18-100A (0.39 x 15 cr column, with a 680 gradient controller and a- 991 diode array detector. An acetonitrile gradient of 0 to 30% (1%/min) in water, and 0.1% trifluoroacetic acid (TPA) at 30 0 C and a flow of 1 ml/min were used. Before being coupled to KLH, the peptides were purified or a 2 x 25 cm C18 Bischoff Prap 2025 column in Hewlett Packard 1082B HPLC equipment and with a reading at 225 nm. Methanol gradients with a methanol increase of 1% per min at a flow of 10 ml/min in water and 0.1% of trifluoroacetic acid (TFA) were used. Depending on the peptide, the initial methanol concentrations varied between 0 and 20%. The amino acid composition analysis was carried out in accordance with the Pico-tag (Waters) processes.

t' 9 antibodies which eliminate the vaccine. The data known heretofore point to the fact that among the general population of anti-virus antibodies in infected

Q

Example 2 CHEMICAL

SELECTION

Based upon the data the PPV capsid, the were synthesized.

SYNTHESIS OF PPV PEPTIDES AND OF IMMUNOGENIC PEPTIDES as to the location of B epitopes on seventeen peptides shown in Table 2 Code -7L15 1L15 5L16 6L1 5 7L15 8L15 1OL16 13L15 83L17 22 5L1 5 293L20 346L16 364L15 380L19 408L19 427L18 294L11/87L8 TABLE 2 Sequence

*NTNSNSMSENVEQHN#

*MSENVEQHNPINAGT#

*VEQHNPINAFTELSAT#

EQHNPTNAGTELSAT#

QHNPINAGTELsATG#

*HNPINAGTELSATGN#

*PINAGTELSATGITESG#

*AGTELSATGNESGGG#

IHVLNSESGSAGQMVQD#

GQ SQQ IT DS IQTGLH#

*LTEPTTEGDQHPGTLPAANTC#

YSNGGPFLTPIVPT,D#

YNDDEPNGAIRFT4G#

*QHGHLTTSSQELERYTFNP#

QQFNQQAPLNLENTNNGTL#

*LPSDPIGGKSNMHFMNTL#

*TEPTTEGDQHPNSESGSAG#

It can be seen that of the seventeen peptides synthesized eight are located at the terminal amino region of VP2 in PPV whereas the remaining nine are located at internal VP2 regions in PPV. The length of the peptides varies between 15 and 20 residues, not including the N-terminal cysteine that is used for coupling purposes. They were all sufficiently soluble to allow their conjugation to

KLH.

The synthesis of these 17 peptides was carried out using Emoc cheisiry, pursuant to a general process mentionp!r

A

18

TM

above, on RinkM resins (Example The sequences were obtained from the VP2 protein in PPV (Ranz et al., cited supra; Sakurai et al., Virus Res. 13, 79-86 (1989)). The sequences are acetylated at the N-terminal end and amidated at the C-terminal. Some peptides were purified in accordance with the protocol described in Example 1.

Example 3 CONJUGATING THE PEPTIDES TO KLH Coupling of the purified peptides to the carrier KLH protein (Calbiochem) was carried out in accordance with standard processes using m-maleimidobenzoyl-N-succinimide ester (MBS) as coupling agent (Lerner et al. (1981) Proc.

Natl. Acad. Sci. USA 78, 3403-3407). The dialysis steps were carried out at 4 0 C and the other incubations at room temperature. One mg of peptide was added to every mg of KLH after attachment of a linker to the protein and this I was incubated overnight at room temperature. The conjugates were then stored either directly (conjugates not dialyzed) or dialyzed, with three changes of 0.1M, pH:5.0 sodium phosphate overnight against phosphate buffered saline solution (PBS). The conjugates were divided into aliquot parts containing 1 mg of KLH and were stored at -20 0 C. The ratios of gg of peptide per mg of KLH varied between 82 and 304, depending upon the peptide used.

Example 4 IMMUNIZATION OF RABBITS WITH CPV PEPTIDES New Zealand White rabbits were used for immunizations using peptides with a view to inducing neutralizing antibodies. The peptide-KLH conjugates (not dialyzed) corresponding to 1 mg of peptide in 1 ml of buffer (PBS) were mixed with an equal volume of Freund complete adjuvant (FCA) and were intramuscularly and subcutaneously injv-cted into rabbits. After 65 days the o animals received a second immunization with the same ~I r_ tor atnigenic sites in PPV. The results obtained showed that, as with CPV, the amino terminal end of VP2 in PPV 3 is a potential neutralization site.

19 immunogen mixed with Freund incomplete adjuvant (FIA).

Blood was taken on days 0, 57, 64 and 70. When the titres of anti-peptide antibodies in the peptide ELISA increased by a factor of at least 5 after the second immunization, the animals received a second boost with the same amount of conjugate in FIA on day 72 and blood was taken on days and 102.

4.1 Assessment of the anti-CPV antibody titre in rabbit serum.

4.1.A. Specific virus ELISA The presence of specific anti-CPV antibodies in the serum of the immunized animals was established with an indirect ELISA assay. The antigen used was purified virus.

Briefly, polystyrene plates were coated with 0.5 gg of virus in 100 gl of carbonate buffer (0.05 M, pH 9.6) at overnight. The plates were washed with PBS (0.15M NaCl in 0.1 M sodium phosphate pH 7.4) containing 0.5% Tweenand were incubated with the antiserum for 2h at 37 0

C,

washed again and incubated with anti-rabbit goat IgG labelled with biotin for 1 h at room temperature. They were subsequently incubated with streptavidin labelled with peroxidase for 30 min at room temperature. The plates were washed again and the reaction was developed with o-phenylenediamine (OPD) as a substrate for peroxidase, for 10 min in darkness and read at 450 nm in a lultichannel spectrophotometer.

The results obtained are shown in Table 3, from which it follows that all the rabbits produced anti-CPV antibodies.

4.1.B Specific peptide ELISA An indirect ELISA assay was used to establish the presence of anti-peptide antibodies. Flat bottom polystyrene plates were coated overnight with 1 pg of iHT 4°ovenigt. Te pate wee wahedwit PB (0.5M ad i peptide in a 50 mM sodium carbonate solution pH 9.6 at 4 0 C. The first antibody, rabbit serum diluted in incubatio buffer comprising PBS containing 4% of horse serum, 1% of Tween 80 and NaCl 0.5 M were added. After incubating for lh at 25 0 C, the plates were washed with PBS containing 0.05% of Tween 80. The plates were then incubated with anti-rabbit IgGperoxidase conjugates diluted 1/100 in the incubation buffer, and the plates were washed as before. Finally, the coloured reaction was formed adding the substrate tetramethylbenzidine (Aldrich) in 0.1M sodium acetate pH and 0.035% H 2 0 2 for 20 min at room temperature.

The reaction was stopped adding 50 pl of 1.5M H 2

SO

4 The Sabsorbance was measured at 450 nm.

The results obtained are shown in Table 3 from which it follows that all the rabbits produced antibodies against Sthe peptides used to immunize the -eme.

4.2. Neutralization of CPV "in vitro" Neutralization was established with a monolayer protection test. Briefly, a known amount of CPV (100 units of HA) was incubated with rabbit antiserum at different dilutions for 2 hours at 37 0 C. The samples were then inoculated on monolayers of susceptible CRFK cells (ATCC CCL 94) for 90 min at 37 0 C. The monolayers were coated with 1 ml of 1% agarose in fresh medium. Five days after infection the cells were fixed with formaldehyde in PBS for 20 min, the agarose was removed and the remaining cells were stained with 1% crystal violet in 50% ethanol for 20 min. The level of protection was assessed by visual screening of the infected mono layers. The results obtained are shown in Table 3 where it can be seen that the 1L15, 2L15, 3L15, 4L15, 5L15, 6L15, 7L15, 8L15 and 9L15 were capable of inducing CPV neutralizing l i 1 1 1 the following key: :anti-1L15 titre in sentry dog (3025 serum :anti_-lL5-tit 21 r antibodies in rabbits.

The overall results of the immunization of rabbits with CPV peptides are shown in Table 3 from which it follows that neutralization values of between 1/100 and 1/3200 were obtained. In the light of these results it can be concluded that most of the peptides located between peptides 1L15 and 7L15 induce a response of neutralizing antibodies which is even more effective than the mixture used in the vaccination of dogs. Indeed in absolute terms, the neutralization values obtained are better than the results obtained with naturally infected dog sera.

This result allows it to be concluded that the twentyfive residues composing the amino terminal end of the VP2 in CPV constitute a highly immunogenic region and that peptides comprised therein contain the required antigenic potential to prepare useful synthetic vaccines to prevent i canine, feline and mink parvovirosis. S i i S r"

I

al., Proc. Natl. Acad. Sci., USA, 81:3998-4002 (1984)) ;that there are ten antigenic sites in,,the VP2 of CPV am t ik TABLE 3 ELISA PEPTIDE NEUTRALI ZATION TITRE ANTI-PEPTIDE

REACTIVITY

ANTI -VIRUS R13ACTIVTTY -10L15 5L15 1L15 2L15 3Li15 4L15 5L15 6L15 7L15 8L15 9L15 11L15 15L15 157L17 172L15 283L15 296L15 498L15 549L15 570L15 Ye ote+vhl 1/100 1/1600 1/ 24 00 1/200 1/ 32 00 1/1600 1/500 1/ 1600 1/400 k I~ f Ii

A

Anti-virus ELISA: ±<102. +:1o2-103; 10 4; *:Measured by PEPSCAN (elsewhere by anti-peptide ELISA) at a serum dilution of 1/100, and K> absorbance units (optical density) It can be seen that all the rabbits produced antibodies against the respective peptides use.d in immunization.,The most outstanding result was obtained with the 1Ll5, /2L15

L

I 7_1 23 3L15, 4L15, 5L15, 6L15, 7L15, 8L15 and 9L15 peptides which were capable of inducing neutralizing antibodies, some, 2L15, 3L15, 5L15, 6L15 and 8L15 peptides at levels i similar to those obtained when using complete virus as immunogen (>1:1600). Any of these peptides or combinations thereof can be selected for inducing neutralizing antibodies in dogs and their application as a vaccine.

Example 5 DOG IMMUNIZATION In order to verify the vaccinal effect of peptides on the natural hosts (dogs) an immunization test was made which included the subsequent infection with the same virus.

The test animals used were three SPF (specific pathogen free) Beagle dogs. Two of the dogs (identified as 3029 and 3030) were immunized with a mixture in equal parts of the 1L15 and 7L15 peptides separately conjugated to KLH.

The immunization mixture or cocktail (2.5 ml of total volume) contained 1 mg of each of the peptides coupled to KLH adsorbed on alumina gels and with QuilA adjuvant Mg/dog). The vaccination schedule was as follows: the first dose was given on day 0, and 4 weeks later they were given a record dose. Ten weeks later the virulent virus challenge test was made, lying under observation for a further 2 weeks. The dog used as negative sentry (identified as 3025) received a mixture of buffer (PBS)/ adjuvant without conjugate. The dogs were bled on days 0, 29, 36, 43, 50, 57, 64, 71 and every three days after the challenge until the test was over.

The faeces of a dog that had died as a consequence of a parvovirus infection and which contained virulent virus were used for the challenge. The faeces were homogenized in sterile culture medium and the virus was extracted with chlorqform. This material was applied in oral-nasal swabs on the dogs at the time of infection. The presence ^p 24 of CPV in these faeces had been checked using hemagglutination and cell culture techniques.

The anti-CPV antibody titre in the serum of the dogs was assessed by: a) Specific virus ELISA The protocol described in section 4.1.A above was followed albeit using dog antisera as first antibody and incubating the plates containing such antisera with Protein A labelled with peroxidase for 1 h at room temperature. The reaction was developed with OPD and read at 450 nm in a multichannel spectrophotometer. The results obtained are shown in figure 1 from which it follows that after some 15 days post-lst vaccination, the immunized animal (dogs 3029-A-and 3030-0-) sera contained high anti-CPV antibodies titres, which were relatively constant after the 2nd immunization (4th week).

Subsequently, after the virulent virus challenge test, there was a slight increase in the CPV antibody titre in the immunized dogs, and the sentry dog (dog 3025-3-) died after 6 days.

b) Specific peptide ELISA The protocol described in example 4.1.B was followed though serum was added to the dogs to be assessed diluted in the incubation buffer as first antibody. The plates were subsequently incubated with anti-dog IgG-peroxidase conjugates diluted 1/100 in the incubation buffer and finally the coloured reaction was formed adding tetramethylbenzidine, stoppe, with H 2

SO

4 and absorbance was measured at 450 nm.

The results obtained are shown in figure 2 from which it follows that the immunized dogs (3029 and 3030) produced airtibodies which recognized the 1L15 and (3030 and 7L15 (3029 and (3030 peptides whereas the serum of the negative sentry dog d 'r i

I

1 yi i If ,j 1 1 1 i* i.1...11 lii-~ iiii-i 11 *iii~i~iini:ii« iM M l« ^|iM MC ti''^ (3025) contained no antibodies recognizing the 1L15 and 7L15 peptides. After the virulent virus challenge the sentry dog (3025) died whereas the sera of the immunized dogs increased slightly in their antibody content rn aognizing the 1L15 and 7L15 peptides.

c) "In vitro" neutralization of CPV The protocol described in Example 4.2 was followed but the CPV was incubated with antiserum of the dogs (3025, 3029 and 3030) at different dilutions. After inoculating the samples on monolayers of CRFK cells and continuing the aforesaid protocol the results shown in figure 3 were obtained, from which it follows that the immunized dogs (3029-A-and 3030- produced neutralizing antibodies at levels similar to those obtained using complete virus as immunogen (approximately 1:1000). The sentry dog (3025 however never produced any neutralizing antibodies before the challenge.

d) Establishing CPV in dog faeces The presence of CPV in immunized dog faeces was established using a double antibody sandwich ELISA assay (ELISA-DAS) pursuant to the methodology described by Rimmelzwaan et al. (Vet. Quart., 1990, 12, 14-20). The results obtained are shown in figure 4 from which it follows that the faeces of the immunized dogs (3029--and 3030-G-) contained almost no CPV whereas the faeces of the"' sentry dog (3025 however contained high CPV levels after the virulent virus challenge.

e) Clinical assessment Together with the previously described serological data the progress of the disease was clinically monitored, recording temperature and external signs daily. The animal faeces were also collected daily. Clinically, it r-^ (3025) died after 6 days, showing all characteristic symptoms of parvovirosis (diarrhoea, blood in stools and presence of CPV). The two immunized dogs developed high anti-peptide and anti-CPV antibody titres, including a high neutralizing activity which reached its peak on week (Fig. 3) after the ist immunization. The second dose did not reinforce the antibody titre. The sentry dog was negative over the first vaccination period. After the oral-nasal challenge with CPV, both vaccinated dogs were healthy for the complete period.

Example 6 MINK IMMUNIZATION This mink immunization test was made due to the close homology between CPV, FPLV and MEV in order to experimentally check the effectiveness of these new peptides in the design of MEV vaccines.

6.a Immunization Three groups of minks (genotype A/A, seronegative for the Aleutian disease virus and the mink enteritis virus (MEV)) were used. Group 1 (nos. 1-6) were vaccinate with 0.5 mg of the 1L15 peptide coupled to 0.5 mg of KLH plus 0 mg of the 7Li5 peptide coupled to 0.5 mg of KLH, using 50 pg of ISCOM mitrix as adjuvant, all in a final volume of 1 ml of PBS (pH: Group 2 (nos. 7-12) only received 1 ml of PBS (pH: Group 3 (nos. 13.18) received a conventional vaccine (BIOVAC

TM

United, DK) based upon inactivated virus which was administered following the manufacturer's recommendations. The minks all received a single sibcutaneous vaccinal dose and were bled just before vaccination.

Eape6MN4MUIAIN.-' Thismi~n imuniztio tes wa mad du to he lose*1 I! i: :rr 27 6.b Challenge Blood samples were taken twenty-one days after vaccination and the minks were left fasting for 24 h.

They were then infected by oral-nasal means using 0.8 ml of a 25% intestinal homogenate of minks suffering from acute MEV infection. Fecal samples of all the minks were checked daily to detect MEV, using an ELISA based upon specific rabbit antisera. The animals in group 2 (negat-,ve sentries) secreted massive amounts of virus on days 5, 6 and 7 after the challenge. They were all sacrificed without pain in dying state because of a severe disease.

The two other groups of minks secreted no virus in their faeces. Only one mink in group 1 displayed a small indication of virus on day 6, though all the animals were clinically normal, free from diarrhoea and had a good appetite throughout the test.

The virus secreted by the sentry group was extracted, from the positive fecal samples and underwent immunoelectron microscopy, and the presence of a large number of MEV P, particles was confirmed.

6.c Establishing neutralizing antibodies The sera of the minks were all checked for their neutralizing activity on MEV cultivated in CRFK cells.

The'non-neutralized virus was detected by means of the immtino.iaroxidase technique using microtitre plates containing CRFK cells infected with 100 DICT 50 tissue culture infective dose) of MEV, infected for 4 days and fixed Holm-Jensen, Acta Vet. Scand., 22:85- 98 (1981)). Incubation at 37 0 C for 1 h with an antibody linked to peroxidase and development followed. The titres obtained eRe shown in Table 4. The titre at the final point was stated as the highest inverse serum dilution neutralizing the virus infection on the monolayer of

I

Bi i! 28 cells.

Twenty-one days after vaccination the groups that had received the commercial vaccine showed good neutralization titres 31og10). The sentry animals and the group vaccinated with the pep-cidic vaccine showed no neutralizing antibodies in the "in vitro" assay, which indicates that complete disease protection can be obtained induced by a synthetic peptide without inducing neutralizing antibodies "in vitro". It could be speculated that protection could be due to either the inhibition of a cell attachment site by the antibodies to the peptide or the induction of a mechanism mediated by ccnlls.

Two weeks after the challenge the minks all showed MEV neutralizing antibodies. In conclusion, it can be stated that with a synthetic vaccine based upon the peptides of i this invention solid protection of minks can be obtained, 3j 'and they can hence also be formulated in the preparation i of an effective vaccine.

1 I i .7 ii If i 7 Mink 1 2 3 4 6 7

P

9 11 12 13 14 16 17 18

TABLE

Titre (log10) [21 days postvaccination] <1.0 <1.0 <1.0 <1.0 <1.0 <1.0 <1.0 <1.0 <1.0 <1.0 <1.0 <1.0 >3.1 >3.1 >3.1 >3.1 >3.1 >3.1 Titre (loglo) [14 days postchallenge] 2.7 3.1 >3.1 >3.1 3.1 3.1 >3.1 >3.1 >3.1 >3.1 >3.1 >3.1 >3.1 >3.1 >3.1 >3.1 Example 7 RABBIT IMMUNIZATION WITH PPV PEPTIDES New Zealand White rabbits were used for immunizations with peptides with a view to inducing neutralizing antibodies. The peptide-KLH conjugates corresponding to 1 mg of peptide in 1 ml of buffer (PBS) were mixed with an equal volume of Freund complete adjuvant (FCA) and intramuscularly and subcutaneously injected into rabbits.

After 42 days the animals received a second immunization with the same immunogen mixed with Freund incomplete adjuvant (FIA). Blood was taken on days 0, 39, 50 and 71.

Ki 4 4 7.1 Western Blot (immunoblot) To establish the specificity of the antibodies induced by the peptides in the rabbits a Western Blot assay was used. To this end, complete purified PPV was run in a polyacrylamide-SDS (sodium dodecylsulphate) gel under the usual conditions (Laemli, Nature 227:680-685 (1970)). The proteins were then transferred to PVDF filters (polyvinylydene difluoride) (Millipore, USA) using a semi-dry apparatus (KEM-En-Tec, DK) and immunoblot was carried out pursuant to set techniques (Burnette, Anal. Biochem., 112:195-203 (1981); De Bias D.L. et al., Anal. Biochem., 133:214-219 (1983)).

Briefly, the filters were blocked in a solution containing 2% skim milk for 30 minutes. Anti-PPV rabbit serum, 1:1000 diluted, was then added for 2 h at room temperature and overnight at 50C. This was then washed twice and finally incubated for 2 h with rabbit anti-IgG labelled with peroxidase (Dako P217, DK) 1:500 diluted.

The chromogen used was o-dianisidine. The results showing reactivity with the structural proteins are shown in Table 7.2 Neutralization of PPVI"in vitro" Serial double dilutions of each serum, inactivated at 56 °C for 30 min, were prepared in Eagle's minimum essential medium (Eagle's MEM). Fifty gl of each dilution were then mixed with 100-300 DICT 50 of the PPV Danish strain 893 (SVIV, Lindholm) in 50 pl of Eagle medium and left to incubate for 60 min at 37°C. Each dilution was then mixed in a plate of 96 wells with 2000 primary pig j kidney cells in 50 ml of Eagle's MEM and calf fetal serum up to a final concentration of 7% and was left to incubate for 4-5 days at 37 0

C.

The plates were stained using a monolayer immunoperoxidase assay, using a porcine anti-PPV serum, s1' i i: I i L; ii )i i i j ,i i

I

i i r i i:i.

a 31 anti pig rabbit immunoglobin attached to peroxidase and blotting with amino-ethyl-carbazole, following conventional protocols (Holm-Jensen cited supra) The final point titre was stated as the highest inverse serum dilution neutralizing the virus infection on the mono layer of cells.

The results obtained are shown in Table 5 from which it follows that the 1L,15, 5L16, 6L15, 8L15, 10L16 and 13,15 peptides were capable of inducing antibodies neutralizing PPV in rabbits.

TABLE PEPTIDE ANTISERA WESTERN BLOT NEUTRALIZATION -71,15 5L1 6 6L15 7L15 8L15 1OL16 13Li15 83L15 225L15 293L20 34 6L1 6 364L15 380L19 408L19 427L18 294L11/87L8 Key to the symbols: Neutralization titre: VPi ,VP2 VPl VP2 VPl,VP2 VPl, VP2 VPl ,VP2 VPl,VP2,VP3 VPl 1 VP2 ,VP3 VP1,VP2,VP3 VP1,VP2 ,VP3 VP1,VP2,VP3 VPi ,VP2 ,VP3 VP1,VP2,VP3 VPl,VP2,VP3 VP1,VP2,VP3 VP1,VP2,VP3 2-20.

4, -4 4 IntL or*1 A pilcion No PCT/ES 94/00006 r animals received a second immunization with the same i 32 It is clear that all the rabbits produced antibodies against the PPV capsid proteins, which was established by Western Blot. The most outstanding result was obtained with the 1L15, 5L16, 6L15, 8L15, 10L16 and 13L15 peptides which were capable of inducing antibodies capable of neutralizing PPV "in vitro". These peptides were selected for inducing neutralizing antibodies in pigs and the application thereof as a vaccine.

Example 8 PIG IMMUNIZATION To check the immunogenicity of peptides on the host animal the peptide designated 5L16 (Table 2) was used.

Three PPV seronegative Gbttingen mini-pigs were vaccinated with 1 mg of 5L16 peptide coupled to 1 mg of KLH each. The adjuvant used was ISCOM Matrix (500 gg) and the sample was taken to 2 ml with PBS. The pigs were vaccinated twice with an interval of three weeks. The serum samples were taken on days 0, 25 and 36.

8.a PPV ELISA Assay PPV virions were adsorbed to flat bottom polystyrene plates (Nunc). Seriated dilutions (2 factor) of the pig serum were then added to the plates and incubated at 37 0

C

for 1 h. The plates were washed and pig anti rabbit-IgG serum conjugated to peroxidase was added. Incubation for min at 379C and OPD blotting followed.

The three mini-pigs failed to show an anti-PPV antibodies titre on, days 0 and 25. Now then, on day 36 (10 days after revaccination) significant albeit low (Table 6) titres had developed, showing that the peptidic vaccine is capable of inducing antibodies in pigs which recognize the epitope in purified PPV virions.

:il

I

presence or anti-peptiae anticooaces. e.±a-t ouo-0 S 35 polystyrene plates were coated overnight with 1 pg of 7/ 33 TABLE 6 Mini-pig no. Day 0 titre Day 25 titre Day 36 titre 284 28 35 130 S283 21 43 226 280 28 31 1194 8.b Western Blot Analysis A Western Blot analysis was made to analyze reactivity and specificity, against the various protometers of the virus, of the sera originating in the three immunized mini-pigs.

The procedure followed to check reactivity by Western Blot was similar to that used in Example 7.1, with slight modifications. The blocking buffer contained 1% casein instead of fetal calf serum and albumin. The conjugate used was pig anti-IgG rabbit serum labelled with peroxidase (Dako P164).

The results showed a very strong reaction against VP1 and VP2 after the two immunizations. These results confirm the high immunogenicity of these peptides, which confirms that they are useful in formulating new PPV subunit vaccines.

Example 9 FORMULATING A VACCINE Vaccines capable of protecting dogs, cats, minks and pigs respectively from CPV, FPLV, MEV and PPV infection containing one or more of the corresponding peptides provided by this invention coupled to a carrier protein or multimeric structure, or in the form of an immunogenic complex, or in the form of a recombinant immunogenic protein, with an immunologically acceptable diluent such as a physiological pH buffered saline solution, plus an adjuvant such as Alhydrogel in combination with QuilA gg/animal) can be prepared. A single injection can be sufficient to confer animals with protection from the 34 disease though, in some cases, depending upon the assessment of the antibody titre, it could be useful to use a second booster dose.

Translation of the keys to the figures Figures 1 and 3 titre logarithm days sentry dog (3025) vaccinated dog (3029) vaccinated dog (3030) 1st immunization 2nd immunization virulent virus challenge Figure 2 titre logarithm days dog 3025, peptide 1L15 dog 3029, peptide 1L15 dog 3030, peptide 1L15 dog 3025, peptide 7L15 dog 3029, peptide 7L15 dog 3030, peptide 7L15 1st immunization 2nd immunization virulent virus challenge Figure 4 Absorbance x 1000 (450 nm) d ys sentry dog (3025) vaccinated dog (3029) vaccinated dog (3030) 1st immunization 2nd immunization virulent virus challenge I 9. T

:J!J

SEQUENCE LIST INFORMATION ON IDENTIFIED SEQUENCE No. 1: i) SEQUENCE CHARACTERISTICS: LENGTH: 25 Amino Acids TYPE: Amino Acids TOPOLOGY: Linear ii) MOLECULE TYPE: Peptide v) FRAGMENT TYPE: N-terminal vi) ORIGINAL SOURCE: ORGANISM: Canine Parvovirus (CPV) STRAIN: Type 2 Xi) SEQUENCE DESCRIPTION: ID. SEQ. No.: 1 Met Ser Asp Gly Ala Val Gin Pro Asp Gly Gly Gin Pro Ala 1 5 Val Arg Asn Glu Arg Ala Thr Gly Ser Gly Asn 20 iI 'i 3 4* against the respective peptides used in immunization. The most outstanding result was obtained with the 1L15, /2L15, i; INFORMATION ON IDENTIFIED SEQUENCE No. 2: i) SEQUENCE CHARACTERISTICS: LENGTH: 25 Amino Acids TYPE: Amino Acids TOPOLOGY: Linear ii) MOLECULE TYPE: Peptide v) FRAGMENT TYPE: N-terminal vi) ORIGINAL SOURCE: ORGANISM: Porcine Parvovirus (PPV) .0 STRAIN: NADL-2 xi) SEQUENCE DESCRIPTION: ID. SEQ. No.: 2 Met Ser Glu Asn Val Glu Gin His Asn Pro Ile Asn Ala Gly 1 Thr 15 Glu Leu Ser Ala Thr Gly Asn Glu Ser Gly 20 25

S,

'1 i

Claims

1. An immunogenic peptide comprising aH contiguous sequence of 6 to 25 amino acids selected from the group consisting of: a) the sequence shown in ID. SEQ. No. 1; and b) the sequence shown in ID. SEQ. No. 2.

2. A peptide according to claim 1, wherein said contiguouis sequence comprises 15 to 20 amino acids.

3. A peptide according to Claims 1 or 2, wherein said peptide is a chemically synthesized peptide. *55*4. A peptide accordiag to claim 1, selected from the group consisting of: Met-Ser-Asp-Gly-Ala-Val -Gln-Pro-Asp-Gly-Gly--Gln-Pro-Ala 20 Se -s -l -l -a -l -r -s -l -l -l -r -l -a -Arg; Gly-Ala-Val -Gln-Pro-Asp-Gly-Gly-Gln-Pro-Ala-Val -Arg--Asn I -Gl; a-l-PoApGy-l-l-roAaVlAr-s-l -Arg; Val -Gln-Pro-Asp-Gly-Gly-Gln--Pro-Ala-Val -Arg-Asn-Glu-Arg -Ala; -Thr; INOREDEI-QEA NTRACOAL4 SOH Intrnmo~l-N PCT/S 940000 OCUMNTOSCONSDEILDOS ERTIENTE I Ientfii-b- o Z i t__M r~~7 4 S# C$t. C I

559. C 9 CItt CC C C C CCC Pro-Asp-Gly-Gly-Gln-Pro-Ala-Va>Arg-Asn-GluArg.Ala-Thr -Gly; y Asp-Gly-Gly-Gln--Pro-Ala-Val -Arg-Asn-Glu-Arg-Ala-Thr-.Gly -Ser. A peptide according to Claim 4, wherein said peptide is a chemically synthesized peptide. 6. A pe 'tide according to claims 4 or 5, wherein said peptide is capable of inducing neutralizing antibodies to canine parvovirus (CPV). 7. A peptide according to claim 1, selected from the group consisting of: Met -Ser--Asp-Gly-Ala-Val -Gln-Pro-Asp-Gly-Gly-Glnt-Pro-Ala -Val; and Gl r -s -l l -l -r -l -a -r -s -l -r -l Thr. 8. A peptide according to Claim 7, wherein said peptide is a chemically synthesized peptide. 9. A peptide according to claims 7 or 8, wherein said peptide is capaj')le of inducing neutralizing antibodies to mink enteritis virus (MEv). A peptide according to claim 1, selected from the group consisting of: Met,-Ser-Glu-Asn-Val-Glu-Gln-His-Asn-Pro Ile-Asn-Ala-Gly Thr;, C C C t-C~ CCC. It CC CC I S 5* 9 I. 9 0 39 Val -Glu-Gln-His-Asn-Pro- Ile-Asn-Ala-Gly-Thr-Glu-Leu-Ser -Ala--Thr; Glu-Gln-His-Asn-Pro-Ile-Asn-Ala-Gly-Thr-Glu-Leu-Ser-Ala -Thr; Gln-His-Asn-Pro-Ile--Asn-Ala-Gly-Thr-Glu-Leu-Ser-Ala-Thr -Gly; His-Asn-Pro-Ile--Asn-Ala-Gly-Thr-Glu-Leu-Ser-Ala-Thr-Gly -Asn; Pro- Ile-Asn-Ala-Gly-Thr-Glu-Leu-Ser-Ala-Thr-Gly-Asn-Glu V -Ser-Gly; and Ala-Gly-Thr-Glu-Leu-Ser-Ala-Thr-Gly-Asn-Glu-Ser-Gly-Gly 11. A peptide according to Claim 10, wherein said 4So 20 peptide is a chemically synthesized peptide. 12. A peptie according to claims 10 or 11, wherein said peptide is capable of inducing neutralizing antibodies to porcine parvovirus (PPV). S 13. A vaccine capable of protecting animals against parvovirus infection, comprising: a) an immunizing amount of one or more peptides according to jany one of claims 1 to 12, optionally coupled to a suitable carrier protein or multimeric structure; and b) a di luent and an adjuvant, being immunologically acceptable. ~c~ti.~,14 A vaccine capable of protecting dogs against 39a infection caused by canine parvovirus (CPV), comprising: a) an immunizing amount of one or more peptides according to claims 4, 5 or 6, optionally coupled to a suitable carrier protein or multimeric structure; and b) a diluent and an adjuvant, being immunologically acceptable. A vaccine capable of protecting cats against infection caused by feline panleukopenia virus (FPLV), comprising: a) an immunizing amount of one or more peptides according to claims 4, 5 or 6, optionally coupled to a Ssuitable carrier protein or multimeric structure; and 15 b) a diluent and an adjuvant, being Ito* immunologically acceptable. ot 16. A vaccine capable of protecting minks against infection caused by mink enteritis virus (MEV), o 20 comprising: a) an immunizing amount of one or more peptides according to claims 7, 8 or 9, optionally coupled to a suitable carrier protein or multimeric structure; and b) a diluent and an adjuvant, being S 25 immunologically acceptable. 17. A vaccine capable of protecting pigs against infection caused by porcine parvovirus (PPV), S comprising: a) an immunizing amount of one or more peptides according to claims 10, 11 or 12, optionally coupled to a suitable carrier protein or multimeric structure; and b) a dilunt and an adjuvant, being immunologically acceptable.